In 2012, we continued to delete additional secreted proteases from B. anthracis. The strain BH460, created in 2011, and deleted of six proteases, has proven useful in the preparation of a number of recombinant proteins. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). For general use as a host for the production of recombinant proteins, the strain was cured of pXO1 and made permanently sporulation deficient. As an example, BH460 was used to produce recombinant EF, which previously was difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or E. coli hosts. At the beginning of 2012, we analyzed culture supernatants of strain BH460 by mass spectrometry (MS) to identify proteases that continue to be produced that might degrade valuable endogenous proteins (anthrax toxin proteins that are vaccine candidates) or heterologous proteins. Two additional proteases were identified (GBAA_1995 cysteine protease CysP1, and GBAA_4584 minor extracellular protease VpR). These were genetically deleted, producing strain BH480, which lacks 8 proteases. A new Saccharomyces cerevisiae Flp-FRT recombinase system was created and used in B. anthracis for deletion of the cysP1 and vpR genes. The Flp-FRT method replaced the cysP1 and vpR genes with 48-bp FRTsequences as a result of action of the cloned Flp recombinase. Insertion of the FRT sequence avoided potential problems associated with the presence of extra loxP sites remaining in BH460 due to deletion of other proteases. MS analysis confirmed the absence of both proteases in the secretome of the B. anthracis BH480 strain. BH480 is therefore recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture. Work is underway to obtain intellectual property protection and to license this strain to several commercial entities. In a separate project, we continued analyses of the genes and sequences that are needed for maintenance of the key virulence plasmid pXO1, which encodes all three anthrax toxin proteins. In previous work we identified and isolated the region of pXO1 that is responsible for plasmid replication, and thereby defined a minireplicon that was different from one reported by others. It has now become clear through the work of others that the minireplicon we defined is universally conserved among the large family of similar bacillus species plasmids, validating our identification. However, DNA replication alone does not assure that every daughter cell receives a copy of pXO1 when cell division occurs. Thus, we seek to understand the mechanism that causes accurate segregation of plasmids between daughter cells, an active process that assures plasmid maintenance in the bacterial population. Decreased segregational stability of the pXO1 minireplicon in comparison with the native pXO1 was observed in B. anthracis during repeated passage at 37C. This is likely to result from the absence of a functional plasmid maintenance system within the minireplicon region. During the current reporting period of 2012, we applied the new S. cerevisiae Flp-FRT recombination system to the genetic manipulation of B. anthracis. Using this system along with the Cre-loxP system, we identified two regions of pXO1 that are each able to cause plasmid retention. One of these regions lies within pXO1 ORFs 0069-0084. This region is different from the maintenance system reported previously for the large pBtoxis in B. thuringiensis, which has some similarities to pXO1. We found that in this large region only the two genes GBAA_pXO1_0069 and GBAA_pXO1_0084 are essential for pXO1 maintenance. This knowledge provides targets for anti-infective agents - those that do not directly kill the pathogen but instead render it less virulent, thereby allowing host immune responses to effectively combat it. In the year of 2012, we used our gene targeting tools to characterize the function of B. anthracis genes suspected of playing key roles in pathogenesis. We created a B. anthracis mutant strain altered in lipoproteins by deleting the lgt gene encoding the enzyme prolipoprotein diacylglyceryl transferase, which attaches the lipid anchor to prolipoproteins. The in situ complementation method was developed as a second method for confirming the authenticity of the mutation. This method allowed complementation to be done without having to maintain a plasmid under selective pressure. The study of the mutant and complemented strains indicated that lipoprotein biosynthesis in B. anthracis is required for full virulence in a murine infection model, largely due to the importance of lipoproteins present in spores that are needed to sense small host tissue molecules that induce the spores to germinate (i.e., germinants). During this reporting year, we also addressed the issue that B. anthracis cannot be transformed by DNA containing methylated adenosine and cytosine. To identify genes that cause this DNA restriction, we mutated several restriction enzyme genes by means of the Crelox-based system. Thus, we obtained two double mutants, M3-0 and M3-0, both defective in mrr and mcrB3, and a triple mutant, M3-0-1-2, that is additionally defective in mcrBP. This triple mutant can potentially serve as a preferred host for shuttle vectors that express recombinant proteins, including proteins to be used in vaccines. The Cre-loxP genetic modification method was also used to introduce precise genetic knockouts into the B. anthracis gene encoding McsB. The McsB was the only known Tyr kinase reported in B. anthracis. Inactivation of McsB allowed the identification of the first prokaryotic dual specificity tyrosine phosphorylation-regulated kinase PrkD (as described more fully in report AI001031-05). We continued during 2012 to analyze sporulation-deficient mutants of B. anthracis. In the previous reporting period, we showed that B. anthracis frequently generates spontaneous mutants altered in the master sporulation gene spo0A, a signal transduction protein that is essential for bacteria to begin sporulation. In light of the fact that such sporulation mutants were important in identifying the source of the B. anthracis spores used in the 2001 anthrax attacks, we compiled additional data and discussed factors leading to the selection of sporulation mutants, the colony phenotypes these mutants exhibited, and the key role sporulation-impaired mutants played in the FBI Amerithrax investigation. This review article was cited in the 2011 National Academy of Sciences review on the Amerithrax investigation. In 2012, we sent DNA from several additional sporulation-deficient mutants for genomic DNA sequencing so as to identify additional genes that are subject to spontaneous mutation to produce the Spo-negative phenotype.